For the detection of Trypanosoma brucei 18S RNA, we made a pair of biotinylated peptide nucleic acid (PNA) probes which were attached on the surface of fluorescent beads for visualization (). Two fluorescent (strept)avidin coated beads of different size and color [green beads (GB, 1 µm) and red bead (RB, 5.9 µm)] that can easily and simultaneously be located and distinguished from each other by conventional fluorescence microscopy () were chosen. The two biotinylated 18-mer PNA oligomers complementary to and specific for two targets on the Trypanosoma brucei 18S rRNA separated by 191 nucleotides (ca. 60 nm) were bound to the red (PNA3534) and green (PNA3533) beads, respectively ().
Figure 1 Fluorescence microscopy detection of beads and co-localization principle. Red fluorescent beads (RBs) are 5.9 µm in size, surface modified with streptavidin and including magnetite for magnetic separation. Green fluorescent beads (GBs) are 1 µm (more ...)
In a preliminary characterization of this system we chose a simple, short synthetic oligodeoxynucleotide target (instead of RNA). For synthetic reasons the DNA sequence was shortened by129 nt in the middle of the two PNA binding sites (relative to the original parasite 18S RNA target) resulting in a 62 nt DNA. This DNA (97 nt) was sequentially incubated with the pair of fluorescent PNA-beads () for 1 h at room temperature and subjected to fluorescence microscopy analysis. Using varying amounts of DNA (0–1,000 fmol), we consistently found that it is possible to detect co-localization of two beads even at the lowest (40 fmol) concentration, and there was an increase in the number of red and green beads showing co-localization in a dose-dependent manner, although this was not quantitatively addressed in these preliminary experiments (). Furthermore, red beads exhibiting co-localization with more than one green bead were observed at all DNA concentrations, but (as would be expected) appeared more pronounced at higher DNA doses ( and ). To rule out nonspecific complex formation, we performed a number of control experiments (), clearly demonstrating that co-localization requires the presence of DNA and only takes place with PNA coated beads. Furthermore, selective enrichment of the red (magnetic) beads through a washing step makes identification of the double bound target (co-localization) much more effective (). Identification of genuinely hybridization connected (co-localized) green and red beads may be distinguished from coincidental co-localization by difference in vertical location by varying the microscopic focus level, since out of focus beads appear blurred and enlarged (). Nonetheless, we found it advantageous to include a washing step in further experiments for easy detection.
Figure 2 Detection of a 97 nt target DNA with a pair of PNA-Beads. Two PNA beads (red beads (RB) with PNA3534 and green beads (GB) with PNA3533), prepared as illustrated in , were incubated with the target DNA and subsequently analyzed by fluorescence (more ...)
Figure 3 Example of two bead co-localization. (A) Typical examples of co-localization of GB and RB with different GB numbers (e.g., 1, 2 and >3). (B) Detection of GBs at different depth and proximity from RB. Three pictures were taken with different focal (more ...)
To explore the detection sensitivity, we tested this assay with lower DNA doses (200, 40, 8 and 1.6 fmol) (). In analogy with the experiment in , we observed complex formation in a dose-dependent manner, although rigorous quantification was not performed. The detection limit for the target DNA under these conditions was 8 fmol, and as a control for specificity no co-localization was observed using human 18S rRNA, which does not contain targets for the PNAs. In contrast, when we used in vitro transcribed 18S rRNA from the parasite Trypanosoma brucei
(), co-localization of red beads with at least one green bead was observed down to 1.6 fmol RNA, and at concentrations higher than 8 fmol, more than 20% of the red beads showed co-localization with green bead(s) while this number was only around 5% in case of 1.6 fmol RNA (ca 200 beads counted for each). For diagnostic purposes a lower detection limit of 1.6 fmol target RNA is relevant, although it is still one order of magnitude higher than a desired detection limit of 0.16 fmol corresponding to 100 parasites17
given that one parasite contains approximately 1 million copies of 18S-rRNA/parasite.15
Dose dependent detection of target nucleic acids with human 18S rRNA negative control. Experiment analogous to the one presented in .
Figure 5 Dose dependent detection of trypanosoma18S rRNA. The PNA beads (red beads (RB) with PNA3534 and green beads (GB) with PNA3533), were incubated with the indicated amounts of (A) in vitro transcribed target 18S RNA or (B) total trypanosome RNA, and subjected (more ...)
To further validate this assay, we decided to test a total RNA preparation from Trypanosoma brucei
procyclic cultures () in stead of pure target RNA prepared by in vitro transcription. By using this RNA preparation, we were still able to detect co-localization of beads (as indicator for specific hybridization of both PNA probes) down to 1.6 ng of RNA per sample giving >5% of the red beads exhibiting co-localization with at least one green bead. The total RNA amount of 1.6 ng corresponds to ca 0.5 fmol of 18S rRNA (assuming that the 18S rRNA constitutes 20% of the total RNA) and this amount of target rRNA may be obtained from 300 parasites (based on the assumption of 1 million copies of 18S rRNA/cell). For comparison, other methods, including amplification protocols such as real time PCR and loop-mediated isothermal amplification (LAMP)18,19
e.g., in a dipstick format,20
claim to detect infections with 5–10 parasites/ml blood (clinical samples). Although, this sensitivity level has not yet been reached by the presently described bead co-localization principle, a detection limit of 0.5 fmol rRNA is quite encouraging as it is accomplished with a very simple method not requiring amplification. The bead co-localization methodology is indeed a single molecule detection principle and therefore we are confident that it may be significantly optimized in terms of sample preparation and analysis, and should be useful for specific rRNA (or other nucleic acid) detection in general in other contexts, not least within diagnostics of other infectious diseases. The presently used PNAs are specitic for Trypanosoma brucei
, but it remains to be seen to which extent the assay is capable of distinguishing different Trypanosoma species. Furthermore, improved detection limits should be possible if combined with computerized image analysis or by flow cytometric (FACS) or microfluidics sorting for counting co-localized beads.